Structural and Mutagenic Analysis of Foot-and-Mouth Disease Virus 3C Protease Reveals the Role of the β-Ribbon in Proteolysis

Abstract

The 3C protease (3C pro ) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C pro , a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a ␤-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This ␤-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the ␤-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C pro with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the ␤-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C pro inhibitors.

Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity

“…Two synthetic MCoTI-II analogues were reported to exhibit significant activity against 3C protease, a cysteine protease involved in the formation of proteins required for the RNA replication of the foot-and-mouth-disease virus (FMDV) (50,51). This report not only discovered the first peptide-based inhibitor of this agriculturally important viral enzyme, but also demonstrated that the enzyme specificity of this class of cyclotides can be redirected with simple point mutations.…”

“…The 95% confidence intervals (95% CI) were also determined. The relative activity of lysine mutants was estimated using the IC 50 value of kalata B1 divided by the IC 50 value of mutants individually.…”

Three-dimensional structure of foot-and-mouth disease virus and its biological functions

“…The active site of Lb pro with catalytic residues (Asn46, Cys51, His148, and Asp163) and some other important residues (Asn54, Gly145-His148, Asp163-Asp166) contributing to conformational changes is highlighted. (B) Structure of FMDV 3C pro C163A and the active site of the enzyme (PDB:2J92) [134]. The secondary structure of 3C pro is colored as in A.…”

Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues

“…pro , HRV 3C pro , and CVB 3C pro (14,18,20), but was not found in picornaviral proteases with Cys-His-Asp triads (4,5,7,21), suggesting that the hydrogen bond donated by residue Asn69 serves to further stabilize the conformation of the relatively longer glutamic acid side chain, which tends to be more flexible than the aspartic acid counterpart in Cys-His-Asp triads. However, the residue Asn69 is not strictly conserved in picornaviral 3C proteases even in PEV 3C pro , BEV 3C pro , and HRV 3C pro from a different HRV serotype (17) (Fig.…”